During my postdoctoral training at Caltech, I isolated the gene corresponding to the Drosophila shibire mutation. Temperature sensitive shibire mutants become paralyzed, because they fail to reform synaptic vesicles after releasing their contents into the synaptic cleft. The shibire gene was shown to be homologous to dynamin, a protein believed to be a molecular motor. Our work was important because it linked shibire/dynamin to endocytosis in vivo. Using assays for endocytosis in cultured cells, we discovered that dynamin functions after coat proteins assemble at the plasma membrane, but before coated pits become deeply invaginated. Dynamin has many interactions (with microtubules, SH3 domains and acid phospholipids) and proposed functions, but its participation in early stages of endocytosis is the only one that has gained general acceptance. We now propose to continue our studies of the shibire/ dynamin molecule in the nematode C. elegans, which gives a unique opportunity to study dynamin function in vivo. Not only was it easy to obtain mutants, but classic genetics (suppressor screens) and reverse genetics (microinjection of DNA) are straightforward and fast. The specific aims of this proposal are: 1. To Learn More About Dynamin Function by Phenotypic and Expression Analysis. We will determine which of the 302 neurons in C. elegans hermaphrodites express dynamin at high levels. Knowing their function will help understand the mutant phenotypes. We will analyze the phenotype of the dynamin mutants using chromosomal mutations and transgenic techniques. 2. To Characterize the Domain Structure of Dynamin using Reverse Genetics. We will identify functional domains by reverse genetics (easy in C. elegans). We will concentrate on two questions: (l) Which sequences are required for dominant interference? (2) Which domains cause dimerization as inferred from interallelic complementation? 3. To Identify Interacting Proteins through Genetic Analysis. We will identify new molecules involved in synaptic endocytosis using mutant dynamin as a starting point for C. elegans genetics. New genes will be sought by isolating genetic suppressors. Putative interacting proteins will be tested for their ability to bind to dynamin in vitro and in vivo. The identification of new interacting genes will help to understand the molecular events that regulate endocytosis. The use of C. elegans allows us to focus on proteins that are important for synaptic vesicle recycling.